There are many cases when an enzyme protein gene is expressed in a heterologous expression system, the protein is not expressed as an active-form enzyme and this is the biggest problem in a large-scale expression of enzyme protein by a heterologous host. In order to solve the problem, various approaches have been taken. For example, in order to express an active-form enzyme in a host Escherichia coli, cell culture conditions are examined (PTL 1), and the following methods are mentioned: methods in which proteins are co-expressed with molecular chaperones which allow formation of correct conformation (PTL 2), methods in which proteins are expressed as fusion proteins with signal peptides or tags that improve solubility (PTL 3 and PTL 4) and methods in which proteins which form inclusion bodies are unfolded with a denaturing agent and the like and refolded into correct conformation (PTL 5, PTL 6 and PTL 7). In addition, methods in which, without using E. coli, enzyme genes having the same amino acid sequences as the wild type are expressed in yeast, insect or animal cultured cells (NPL 1 and NPL 2) and cell-free translation systems in which entire transcription to translation of genes is carried out in vivo (PTL 8) have also been developed.
However, there are many proteins for which inclusion bodies are not eliminated by co-expression with chaperone genes and other methods also have issues such as complicated procedures and high cost. Therefore, there is still a need for a new technique which allows expression of an enzyme gene as an active-form enzyme by the simplest and the most cost effective way.
Until now, a reported example of expression of a heterologous enzyme in E. coli as an active-form mutant enzyme is expression of a soluble protein from a plant-derived hydroxynitrile lyase gene containing random mutations (PTL 9). There is also a report on a method in which a gene sequence of a target protein is subjected to random mutation followed by the addition of a reporter protein so as to select a sequence that is expressed as an active-form mutant enzyme (NPL 3). However, there is no report on a method that allows specifying a mutation site of a gene or a mutated amino acid that allows expression of a protein as an active-form mutant enzyme.
When genes of various proteins including enzymes are expressed in heterologous expression systems, the proteins are not always expressed as soluble proteins. This is a problem in a large-scale expression of various proteins by a heterologous host. In order to solve the problem, there have been known co-expression with molecular chaperone proteins (NPL 4), expression as fusion proteins with maltose-binding proteins (NPL 5) and the like.